Probing slow timescale dynamics in proteins using methyl <Superscript>1</Superscript>H CEST

Although ¹⁵N- and ¹³C-based chemical exchange saturation transfer (CEST) experiments have assumed an important role in studies of biomolecular conformational exchange, ¹H CEST experiments are only beginning to emerge. We present a methyl-TROSY ¹H CEST experiment that eliminates deleterious ¹H–¹H NOE dips so that CEST profiles can be analyzed robustly to extract methyl proton chemical shifts of rare protein conformers. The utility of the experiment, along with a version that is optimized for ¹³CHD₂ labeled proteins, is established through studies of exchanging protein systems. A comparison between methyl ¹H CEST and methyl ¹H CPMG approaches is presented to highlight the complementarity of the two experiments.     

A new class of CEST experiment based on selecting different magnetization components at the start and end of the CEST relaxation element: an application to <Superscript>1</Superscript>H CEST

Chemical exchange saturation transfer (CEST) experiments are becoming increasingly popular for investigating biomolecular exchange dynamics with rates on the order of approximately 50–500 s^?1 and a rich toolkit of different methods has emerged over the past few years. Typically, experiments are based on the evolution of longitudinal magnetization, or in some cases two-spin order, during a fixed CEST relaxation delay, with the same class of magnetization prepared at the start and selected at end of the CEST period. Here we present a pair of TROSY-based pulse schemes for recording amide and methyl ¹H CEST profiles where longitudinal magnetization at the start evolves to produce two-spin order that is then selected at the completion of the CEST element. This selection process subtracts out contributions from ¹H–¹H cross-relaxation on the fly that would otherwise complicate analysis of the data. It also obviates the need to record spin-state selective CEST profiles as an alternative to eliminating NOE effects, leading to significant improvements in sensitivity. The utility of the approach is demonstrated on a sample of a cavity mutant of T4 lysozyme that undergoes chemical exchange between conformations where the cavity is free and occupied.     

Characterization of the interaction interface and conformational dynamics of human TGIF1 homeodomain upon the binding of consensus DNA

The TG interacting factor-1 homeodomain (TGIF1-HD) binds with the consensus DNA motif 5′-TGTCA-3′ in gene promoters through its three-amino acid loop extension (TALE) type homeodomain, and then recruits co-regulators to regulate gene expression. Although the solution NMR structure of human TGIF1-HD has been reported previously, little is known about its DNA binding mechanism. NMR titrations have been extensively used to study mechanisms of ligand binding to target proteins; however, an intermediate exchange occurred predominantly between TGIF1-HD in the free and bound states when titrated with the consensus DNA, which resulted in poor-quality NMR spectra and precluded further exploration of its interaction interface and conformational dynamics. Here, the helix α3 of TGIF1-HD was speculated as the specific DNA binding interface by hydrogen–deuterium exchange mass spectrometry (HDX-MS) experiments, and subsequently confirmed by chemical exchange saturation transfer (CEST) spectroscopy. In addition, simultaneous conformational changes in other regions, including α1 and α2, were induced by DNA binding, explaining the observation of chemical shift perturbations from extensive residues besides those located in α3. Further, low-populated DNA-bound TGIF1-HD undergoing a slow exchange at a rate of 130.2 ± 3.6 s⁻¹ was derived from the analysis of the CEST data, and two residues, R220 and R221, located in the middle of α3 were identified to be crucial for DNA binding. Our study provides structural and dynamic insights into the mechanisms of TGIF1-HD recognition of extensive promoter DNA.     

Multiple frequency saturation pulses reduce CEST acquisition time for quantifying conformational exchange in biomolecules

Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to ¹H, ¹⁵N or ¹³C. These pseudo 3D datasets are time consuming to collect and are further lengthened by reduced signal to noise stemming from the long saturation pulse. In this article, we show how usage of a multiple frequency saturation pulse (i.e., MF-CEST) changes the nature of data collection from series to parallel, and thus decreases the total acquisition time by an integer factor corresponding to the number of frequencies in the pulse. We demonstrate the applicability of MF-CEST on a Src homology 2 (SH2) domain from phospholipase Cγ and the secondary active transport protein EmrE as model systems by collecting ¹³C methyl and ¹⁵N backbone datasets. MF-CEST can also be extended to additional sites within proteins and nucleic acids. The only notable drawback of MF-CEST as applied to backbone ¹⁵N experiments occurs when a large chemical shift difference between the major and minor populations is present (typically greater than ~?8 ppm). In these cases, ambiguity may arise between the chemical shift of the minor population and the multiple frequency saturation pulse. Nevertheless, this drawback does not occur for methyl group MF-CEST experiments or in cases where somewhat smaller chemical shift differences occur are present.     

Solution NMR and computer simulation studies of active site loop motion in triosephosphate isomerase

Solution NMR spin relaxation experiments and classical MD simulations are used to study the dynamics of triosephosphate isomerase (TIM) in complex with glycerol 3-phosphate (G3P). Three regions in TIM exhibit conformational transitions on the micros-ms time scale as detected by chemical exchange broadening effects in NMR spectroscopy: residue Lys 84 on helix C, located at the dimeric interface; active site loop 6; and helix G. The results indicate that the conformational exchange process affecting the residues of loop 6 is the correlated opening and closing of the loop. Distinct processes are responsible for the chemical exchange linebroadening observed in the other regions of TIM. MD simulations confirm that motions of individual residues within the active site loop are correlated and suggest that the chemical exchange processes observed for residues in helix G arise from transitions between 3(10)- and alpha-helical structures. The results of the joint NMR and MD study provide global insight into the role of conformational dynamic processes in the function of TIM.     

The role of dynamics in allosteric regulation

The biomolecular conformational changes often associated with allostery are, by definition, dynamic processes. Recent publications have disclosed the role of pre-existing equilibria of conformational substates in this process. In addition, the role of dynamics as an entropic carrier of free energy of allostery has been investigated. Recent work thus shows that dynamics is pivotal to allostery, and that it constitutes much more than just the move from the 'T'-state to the 'R'-state. Emerging computational studies have described the actual pathways of allosteric change.     

Monitoring conformational dynamics with solid-state R 1ρ experiments

A new application of solid-state rotating frame (R _1ρ) relaxation experiments to observe conformational dynamics is presented. Studies on a model compound, dimethyl sulfone (DMS), show that R _1ρ relaxation due to reorientation of a chemical shift anisotropy (CSA) tensor undergoing chemical exchange can be used to monitor slow-to-intermediate timescale conformational exchange processes. Control experiments used d ₆ -DMS and alanine to confirm that the technique is monitoring reorientation of the CSA tensor rather than dipolar interactions or methyl group rotation. The application of this method to proteins could represent a new site-specific probe of conformational dynamics.     

Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin

Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm “dynamics govern specificity” might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.     

Protocol for MM/PBSA molecular dynamics simulations of proteins

Continuum solvent models have been employed in past years for understanding processes such as protein folding or biomolecular association. In the last decade, several attempts have been made to merge atomic detail molecular dynamics simulations with solvent continuum models. Among continuum models, the Poisson-Boltzmann solvent accessible surface area model is one of the oldest and most fundamental. Notwithstanding its wide usage for simulation of biomolecular electrostatic potential, the Poisson-Boltzmann equation has been very seldom used to obtain solvation forces for molecular dynamics simulation. We propose here a fast and reliable methodology to implement continuum forces in standard molecular mechanics and dynamics algorithms. Results for a totally unrestrained 1 ns molecular dynamics simulation of a small protein are quantitatively similar to results obtained by explicit solvent molecular dynamics simulations.     

Imaging in vivo extracellular pH with a single paramagnetic chemical exchange saturation transfer magnetic resonance imaging contrast agent

The measurement of extracellular pH (pHe) has potential utility for cancer diagnoses and for assessing the therapeutic effects of pH-dependent therapies. A single magnetic resonance imaging (MRI) contrast agent that is detected through paramagnetic chemical exchange saturation transfer (PARACEST) was designed to measure tumor pH(e) throughout the range of physiologic pH and with magnetic resonance saturation powers that are not harmful to a mouse model of cancer. The chemical characterization and modeling of the contrast agent Yb(3+)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-o-aminoanilide (Yb-DO3A-oAA) suggested that the aryl amine of the agent forms an intramolecular hydrogen bond with a proximal carboxylate ligand, which was essential for generating a practical chemical exchange saturation transfer (CEST) effect from an amine. A ratio of CEST effects from the aryl amine and amide was linearly correlated with pH throughout the physiologic pH range. The pH calibration was used to produce a parametric pH map of a subcutaneous flank tumor on a mouse model of MCF-7 mammary carcinoma. Although refinements in the in vivo CEST MRI methodology may improve the accuracy of pHe measurements, this study demonstrated that the PARACEST contrast agent can be used to generate parametric pH maps of in vivo tumors with saturation power levels that are not harmful to a mouse model of cancer.     

Characterizing and controlling the inherent dynamics of cyclophilin-A

With the recent advances in NMR relaxation techniques, protein motions on functionally important timescales can be studied at atomic resolution. Here, we have used NMR-based relaxation experiments at several temperatures and both 600 and 900 MHz to characterize the inherent dynamics of the enzyme cyclophilin-A (CypA). We have discovered multiple chemical exchange processes within the enzyme that form a “dynamic continuum” that spans 20–30 Å comprising active site residues and residues proximal to the active site. By combining mutagenesis with these NMR relaxation techniques, a simple method of counting the dynamically sampled conformations has been developed. Surprisingly, a combination of point mutations has allowed for the specific regulation of many of the exchange processes that occur within CypA, suggesting that the dynamics of an enzyme may be engineered.     

Charge effects on the fibril-forming peptide KTVIIE: a two-dimensional replica exchange simulation study

The assembly of peptides into ordered nanostructures is increasingly recognized as both a bioengineering tool for generating new materials and a critical aspect of aggregation processes that underlie neurological diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. There is a major problem in understanding how extremely subtle sequence changes can lead to profound and often unexpected differences in self-assembly behavior. To better delineate the complex interplay of different microscopic driving forces in such cases, we develop a methodology to quantify and compare the propensity of different peptide sequences to form small oligomers during early self-assembly stages. This umbrella-sampling replica exchange molecular dynamics method performs a replica exchange molecular dynamics simulation along peptide association reaction coordinates using umbrella restraints. With this method, we study a set of sequence-similar peptides that differ in net charge: K(+)TVIIE(-), K(+)TVIIE, and (+)K(+)TVIIE. Interestingly, experiments show that only the monovalent peptide, K(+)TVIIE, forms fibrils, whereas the others do not. We examine dimer, trimer, and tetramer formation processes of these peptides, and compute high-accuracy potential of mean force association curves. The potential of mean forces recapitulate a higher stability and equilibrium constant of the fibril-forming peptide, similar to experiment, but reveal that entropic contributions to association free energies can play a surprisingly significant role. The simulations also show behavior reminiscent of experimental aggregate polymorphism, revealed in multiple stable conformational states and association pathways. Our results suggest that sequence changes can have significant effects on self-assembly through not only direct peptide-peptide interactions but conformational entropies and degeneracies as well.     

High performance computing in biology: multimillion atom simulations of nanoscale systems

Computational methods have been used in biology for sequence analysis (bioinformatics), all-atom simulation (molecular dynamics and quantum calculations), and more recently for modeling biological networks (systems biology). Of these three techniques, all-atom simulation is currently the most computationally demanding, in terms of compute load, communication speed, and memory load. Breakthroughs in electrostatic force calculation and dynamic load balancing have enabled molecular dynamics simulations of large biomolecular complexes. Here, we report simulation results for the ribosome, using approximately 2.64 million atoms, the largest all-atom biomolecular simulation published to date. Several other nano-scale systems with different numbers of atoms were studied to measure the performance of the NAMD molecular dynamics simulation program on the Los Alamos National Laboratory Q Machine. We demonstrate that multimillion atom systems represent a 'sweet spot' for the NAMD code on large supercomputers. NAMD displays an unprecedented 85% parallel scaling efficiency for the ribosome system on 1024 CPUs. We also review recent targeted molecular dynamics simulations of the ribosome that prove useful for studying conformational changes of this large biomolecular complex in atomic detail.     

Biomolecular structure refinement using the GROMOS simulation software

For the understanding of cellular processes the molecular structure of biomolecules has to be accurately determined. Initial models can be significantly improved by structure refinement techniques. Here, we present the refinement methods and analysis techniques implemented in the GROMOS software for biomolecular simulation. The methodology and some implementation details of the computation of NMR NOE data, ³ J-couplings and residual dipolar couplings, X-ray scattering intensities from crystals and solutions and neutron scattering intensities used in GROMOS is described and refinement strategies and concepts are discussed using example applications. The GROMOS software allows structure refinement combining different types of experimental data with different types of restraining functions, while using a variety of methods to enhance conformational searching and sampling and the thermodynamically calibrated GROMOS force field for biomolecular simulation.     

Time-resolved methods in Biophysics. 2. Monitoring haem proteins at work with nanosecond laser flash photolysis.

Haem proteins have long been the most studied proteins in biophysics, and have become paradigms for the characterization of fundamental biomolecular processes as ligand binding and regulatory conformational transitions. The presence of the haem prosthetic group, the absorbance spectrum of which has a ligation sensitive region conveniently located in the UV-visible range, has offered a powerful and sensitive tool for the investigation of molecular functions. The central Fe atom is capable of reversibly binding diatomic ligands, including O(2), CO, and NO. The Fe-ligand bond is photolabile, and a reactive unligated state can be transiently generated with a pulsed laser. The photodissociated ligands quickly rebind to the haem and the process can be monitored by transient absorbance methods. The ligand rebinding kinetics reflects protein dynamics and ligand migration within the protein inner cavities. The characterization of these processes was done in the past mainly by low temperature experiments. The use of silica gels to trap proteins allows the characterization of internal ligand dynamics at room temperature. In order to show the potential of the laser flash photolysis techniques, combined with modern numerical analysis methods, we report experiments conducted on two non-symbiotic haemoglobins from Arabidopsis thaliana. The comparison between time courses recorded on haemoglobins in solution and encapsulated in silica gels allows for the highlighting of different interplays between protein dynamics and ligand migration.     

Longitudinal relaxation optimized amide <Superscript>1</Superscript>H-CEST experiments for studying slow chemical exchange processes in fully protonated proteins

Chemical Exchange Saturation Transfer (CEST) experiments are increasingly used to study slow timescale exchange processes in biomolecules. Although ¹⁵N- and ¹³C-CEST have been the approaches of choice, the development of spin state selective ¹H-CEST pulse sequences that separate the effects of chemical and dipolar exchange [T. Yuwen, A. Sekhar and L. E. Kay, Angew Chem Int Ed Engl 2016 doi:  10.1002/anie.201610759 (Yuwen et al. 2017)] significantly increases the utility of ¹H-based experiments. Pulse schemes have been described previously for studies of highly deuterated proteins. We present here longitudinal-relaxation optimized amide ¹H-CEST experiments for probing chemical exchange in protonated proteins. Applications involving a pair of proteins are presented establishing that accurate ¹H chemical shifts of sparsely populated conformers can be obtained from simple analyses of ¹H-CEST profiles. A discussion of the inherent differences between ¹⁵N-/¹³C- and ¹H-CEST experiments is presented, leading to an optimal strategy for recording ¹H-CEST experiments.     

NMR methods for characterizing microsecond to millisecond dynamics in recognition and catalysis

During the past two years, significant advances have been made in the development of NMR methods for studying biomolecular dynamics on the microsecond to millisecond timescale. Applications of these methods to biologically relevant systems have provided compelling evidence that, in many cases, conformational dynamics on these timescales govern the rates of biomolecular recognition and catalysis.     

Recent excitements in protein NMR: Large proteins and biologically relevant dynamics

The advent of Transverse Relaxation Optimized SpectroscopY (TROSY) and perdeuteration allowed biomolecular NMR spectroscopists to overcome the size limitation barrier (~20 kDa) in de novo structure determination of proteins. The utility of these techniques was immediately demonstrated on large proteins and protein complexes (e.g. GroEL-GroES, ClpP protease, Hsp90-p53, 20S proteasome, etc.). Further, recent methodological developments such as Residual Dipolar Couplings and Paramagnetic Relaxation Enhancement allowed accurate measurement of long-range structural restraints. Additionally, Carr-Purcell-Meiboom-Gill (CPMG), rotating frame relaxation experiments (R₁ρ) and saturation transfer experiments (CEST and DEST) created never-before accessibility to the μs–ms timescale dynamic parameters that led to the deeper understanding of biological processes. Meanwhile, the excitement in the field continued with a series of developments in the fast data acquisition methods allowing rapid structural studies on less stable proteins. This review aims to discuss important developments in the field of biomolecular NMR spectroscopy in the recent past, i.e., in the post TROSY era. These developments not only gave access to the structural studies of large protein assemblies, but also revolutionized tools in the arsenal of today’s biomolecular NMR and point to a bright future of biomolecular NMR spectroscopy.